{"doi":"10.1016/j.ab.2020.113772","title":"Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions","abstract":"Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the \"holdup\" principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities.","journal":"Analytical Biochemistry","year":2020,"id":82704,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":12,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9614,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":426328,"name":"Anne Förster","orcid":"0000-0001-7466-4414","position":1,"is_corresponding":false},{"id":427363,"name":"Khaled Ould Babah","orcid":null,"position":2,"is_corresponding":false},{"id":398319,"name":"Gergő Gógl","orcid":"0000-0002-8597-3711","position":3,"is_corresponding":false},{"id":427364,"name":"Pascal Eberling","orcid":null,"position":4,"is_corresponding":false},{"id":426329,"name":"Camille Kostmann","orcid":"0000-0002-2881-4909","position":5,"is_corresponding":false},{"id":426330,"name":"Rudolf Volkmer","orcid":"0000-0003-4710-1143","position":6,"is_corresponding":false},{"id":427365,"name":"Victor Tapia Mancilla","orcid":null,"position":7,"is_corresponding":false},{"id":426331,"name":"Gilles Travé","orcid":"0000-0002-4160-4121","position":8,"is_corresponding":false},{"id":426332,"name":"Yves Nominé","orcid":"0000-0002-2912-116X","position":9,"is_corresponding":false},{"id":426327,"name":"Anna Bonhoure","orcid":"0000-0002-0624-890X","position":0,"is_corresponding":true}],"reference_count":35,"raw_metadata":null,"created_at":"2026-07-18T21:53:48.608934Z","pmid":"32428443","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}